Recently, DNA sequences of various species have been determined rapidly, and “structural genomics” is recognized as an important research. With respect to a large number of genes selected from a mass of information about genomic sequences, structural genomics aims the systematic determination of three dimensional structures of proteins coded on each gene, and the comprehensive study of the structure/function relationships.
In the research of structural genomics, many types of proteins which are from 30,000 to more than 40,000 in case of human's proteins, can be targets of structural analysis. Therefore, it is necessary to select the target proteins efficiently. At the same time, actual expressions and preparations of the selected targets are required for the large-scale preparation of samples necessary for structural analysis on a milligram scale.
In the past, for the preparation of such samples, the gene engineering methods in which cloned DNAs are introduced into living cells such as E. coli cell have been generally used. However, these kinds of methods can be applied only in the case where exogenous proteins are restricted to molecular species that can pass through the life-support mechanisms of the host cell. On the other hand, the progress in chemical synthesis technology has made it possible to produce peptides consisting of several tens of amino acids automatically. But, limitations of the yield, the secondary reactions, and other factors make it very difficult to produce proteins with large molecular weights.
As for methods for analyzing the three dimensional structural of proteins, X-ray crystallography has been usually used. The recent use of the synchrotron beam has allowed generation of stronger X-rays (compared with other X-ray generators) in addition to allowing a selection of any wavelength. As a result, even in the case where there is only a kind of heavy atom in the proteins, it has become possible to determine three dimensional structures by the use of MAD (multiwavelength anomalous diffraction) method (Hendrickson, W. A., Science, 254, 51-58 (1991)). One benefit MAD provides is that it reduces the time it takes to determine three dimensional structures of proteins.
In case of the determinations of the three dimensional structures of proteins by the use of X-ray crystallography, preparations of heavy atom isomorphous replacement product of the proteins are necessary for determination of the phase, and, in many cases, proteins containing selenomethionine are used as the said heavy atom isomorphous replacement product. Uses of selenomethionine instead of methionine and expressions of the genes in methionine-requiring auxotrophic mutant strains make it possible to introduce selenium into the proteins.
However, expression systems using living cells have the problems that, because of cytotoxicity of selenomethionine, the expression levels of proteins are very low and sufficient substitution rates of selenomethionine cannot be achieved.
To solve these problems, as a protein synthesis method which harmonizes the biological method with the chemical method, and fully utilizes excellent features of organisms, developments of a cell-free protein synthesis system to produce proteins in vitro by using a cell extract are in progress (for example, Science (1988) 242, 1162-1164, JP Patent Kokai Publication JP-A-4-200390). This cell-free protein synthesis system provides that organic translation systems of genetic information are assorted in artificial chambers, and that synthetic systems are reconstructed in order to introduce any amino acids including non-natural types, by the use of designed nucleic acids as templates.
Because the cell-free protein synthesis system requires complicated and multi step manipulations, and because the productivities were very low in the past, the applications of the cell-free protein synthesis systems were limited. A synthesis system (JP Patent Kokai Publication JP-A-2000-175695) using dialysis has led to the practical application of this technology.
However, the problems in the art have not been overcome yet. In case of the synthesis of heavy atom isomorphous replacement product, preparations of materials for protein synthesis and establishment of the best conditions are complicated and difficult. Additionally, there are no reports that X-ray crystallography of proteins produced by the cell free protein synthesis system has been achieved by the use of the MAD method. Therefore, the introduced rates of heavy atoms to proteins has not been considered sufficiently efficient yet.